Splicing of pre-messenger RNAs is essential to the flow of genetic information in eukaryotes. Splicing is performed by the splicesome, a large molecular machine consisting of RNAs and >100 proteins that assembles anew for each splicing reaction. We have used multi-wavelength single-molecule fluorescence microscopy techniques to directly observe in real time the pathways and dynamics of spliceosome assembly and activation on individual pre-mRNA molecules. The observations reveal new features of these process that have implications for the mechanisms of splicing fidelity and splice site selection.